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目的:探讨喉癌Hep-2细胞系中CD133+肿瘤干细胞的自我更新机制。方法:流式细胞仪分选Hep-2细胞系中CD133+肿瘤细胞,MTT法测定CD133+肿瘤细胞的自我更新能力;Western blot及RT-PCR法测定自我更新相关基因mRNA及蛋白的表达情况。结果:分选前CD133+细胞比例为(3.10±0.21)%,流式细胞仪分选后纯度可达(90.20±5.51)%,体外培养及生长曲线显示分选后的CD133+细胞增殖速度明显较CD133-细胞快,二者比较差异有统计学意义(P<0.01);CD133+细胞中抗凋亡基因Fas、c-myc、survivin mRNA及蛋白表达量均高于CD133-细胞;同时Bcl-2/Bax比例在CD133+细胞中显著升高;CD133+细胞中β-catenin、SHH、SMOH及Bmi-1、Gli-1无论在mRNA水平还是蛋白水平均表达上调,PTCH表达下调。结论:CD133+Hep细胞具有强的增殖能力,抗凋亡基因表达上调是其自我更新的物质基础;干细胞相关信号通路Wnt、Hedgehog以及Bmi-l信号通路在CD133+中处于活化状态,靶向这些信号通路,有望有效杀伤喉癌干细胞。
Objective: To investigate the self-renewal mechanism of CD133 + tumor stem cells in laryngeal carcinoma Hep-2 cell line. Methods: CD133 + tumor cells were sorted by flow cytometry in Hep-2 cell line. The self-renewal ability of CD133 + tumor cells was determined by MTT assay. The mRNA and protein expression of self-renewal genes were detected by Western blot and RT-PCR. Results: The percentage of CD133 + cells before sorting was (3.10 ± 0.21)%, the purity of CD133 + cells was (90.20 ± 5.51)% after sorting by flow cytometry, and the proliferation rate of CD133 + cells after sorting was significantly higher than that of CD133 (P <0.01). The mRNA and protein expressions of Fas, c-myc and survivin in CD133 + cells were significantly higher than those in CD133- cells. The expression of Bcl-2 / Bax The percentage of CD133 + cells was significantly increased. The expression of β-catenin, SHH, SMOH, Bmi-1 and Gli-1 in CD133 + cells was up-regulated at both mRNA and protein levels. CONCLUSION: CD133 + Hep cells have strong proliferative capacity and upregulation of anti-apoptotic genes is the material basis of self-renewal. Stem cell-related signaling pathways Wnt, Hedgehog and Bmi-1 are activated in CD133 + Pathway, is expected to effectively kill laryngeal cancer stem cells.